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The Journal of Immunology, 2000, 165: 3917-3922.
Copyright © 2000 by The American Association of Immunologists

Allergy-Associated Polymorphisms of the Fc{epsilon}RIß Subunit Do Not Impact Its Two Amplification Functions

Emmanuel Donnadieu1,*, William O. Cookson{dagger}, Marie-Hélène Jouvin* and Jean-Pierre Kinet2,*

* Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215; and {dagger} Asthma Genetics Group, University of Oxford, Wellcome Trust Center for Human Genetics, Oxford, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Note added in proof.
 References
 
Two variants of the ß-chain of the high affinity IgE receptor Fc{epsilon}RI, I181L-V183L and E237G, have been found associated with allergy. We have previously shown that the ß-chain plays at least two distinct amplifier functions. It amplifies Fc{epsilon}RI surface expression and signaling, resulting in an estimated 12- to 30-fold amplification of downstream events. To test the hypothesis that the I181L-V183L and E237G ß variants may be functionally relevant and could directly contribute to an allergic phenotype, we have evaluated the functional impact of the ß variants on the two amplifier functions of ß. We found that these variants have no direct effect on the ß amplifier functions. However, the possibility remains that these variants are in linkage disequilibrium with other more relevant polymorphisms or are affecting unknown ß-chain functions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Note added in proof.
 References
 
Allergic (atopic) diseases are characterized by a prolonged production of IgE against common and harmless environmental Ags resulting in inflammatory reactions responsible for a wide range of manifestations including rhinitis, conjunctivitis, dermatitis, and asthma. Atopy results from a combination of numerous environmental and genetic factors. The complexity of the genetic factors involved is evident from the numerous loci for which a linkage to atopy has been reported (1, 2, 3). One of these loci, on chromosome 11q13, contains the gene for the ß-chain of the high affinity IgE receptor, Fc{epsilon}RI (4, 5, 6, 7, 8). Initially, this finding was met with skepticism based on methodological concerns (9), and lack of reproducibility in some populations (10, 11, 12, 13, 14, 15, 16, 17, 18). This lack of reproducibility could have been due to genetic heterogeneity among patients, differences in methodology between studies, and to the existence of a maternal pattern of inheritance (5). However, more recently, this linkage has been confirmed in various populations and by other groups (19, 20, 21, 22, 23, 24, 25, 26), including the Collaborative Study on the Genetics of Asthma (27). It is the strongest in individuals with prominent symptoms, but it is also robust to phenotype classification, which represents an important criteria for assessing the validity of a linkage, given the variability of the criteria used to defined atopy among various studies.

Fc{epsilon}RI controls the activation of mast cells and basophils through IgE, and participates in IgE-mediated Ag presentation in monocytes and dendritic cells. Multivalent Ags bind and cross-link IgEs held at the cell surface by Fc{epsilon}RI. Receptor aggregation induces multiple signaling pathways that control diverse effector responses, including secretion of allergic mediators and induction of cytokine gene transcription (including genes for IL-4, IL-6, TNF-{alpha}, and GM-CSF; reviewed in Refs. 28, 29, 30). Therefore, Fc{epsilon}RI is central to the induction and maintenance of an allergic response. In particular, one component of Fc{epsilon}RI plays a critical role in setting the level of cellular response to IgE and Ag, the ß-chain, through its capacity to amplify both cell surface Fc{epsilon}RI expression (31), and Fc{epsilon}RI signaling (32, 33). Human Fc{epsilon}RI exists as two isoforms, a tetramer {alpha}ß{gamma}2 containing the ß-chain, and a trimer {alpha}{gamma}2 lacking the ß-chain. The overall amplification of cellular responses mediated through the {alpha}ß{gamma}2 receptor is 12- to 30-fold compared with responses mediated through the {alpha}{gamma}2 receptor (31). Given the intensity of this effect, anything affecting the ß-chain is potentially of great value in understanding allergic reactions.

Following the description of the linkage between atopy and the ß locus, a search for mutations in Fc{epsilon}RIß associated with atopy was undertaken and has resulted in the discovery of two polymorphisms in the ß coding sequence (34, 35). These findings have been confirmed independently in different populations in some studies (36, 37) but not in others (14, 36, 38); this may be due in part to the difficulty in sequencing this region of the ß gene. The first one, a double mutation representing an isoleucine to leucine change at position 181 combined with a valine to leucine change at position 183, is located in the fourth transmembrane domain of ß (34). Despite their apparent chemical insignificance, these changes could affect the association of ß with the other chains of Fc{epsilon}RI. This hypothesis is based on the observation that a leucine to isoleucine change at a homologous position in the {zeta} chain reduces its interaction with the IgG binding chain of the Fc{gamma}RIII, CD16 (39). Loss of, or reduction in, association of ß with the IgE binding {alpha}-chain and the signaling {gamma}-chains of Fc{epsilon}RI would result in the expression of fewer and less active receptors, and vice versa. The second polymorphism (35), a glutamic acid to glycine change at position 237, is located in the intracellular tail of ß, just downstream of an immunoreceptor tyrosine-based activation motif essential for ß function (28) and could affect Fc{epsilon}RIß function.

Genetic studies of disease-associated polymorphisms are critical in uncovering pathways implicated in the pathogeny of complex diseases. However, these genetic studies have to be complemented by a direct assessment of the effects of the polymorphisms on the corresponding pathway. This is exemplified in hypertension, a common multifactorial disease. Mutations in a few genes have been shown to be directly responsible for the phenotype in rare forms of the disease (40). Despite the fact that these findings apply to only rare cases of the disease, they have contributed substantially to our understanding of the regulation of blood pressure and may provide insights into the mechanisms underlying common forms of hypertension. The need for a direct assessment of the effect of mutations on a phenotype is also emphasized by cases in which disease-associated polymorphisms have no apparent direct effect on the corresponding phenotype. For example, the allergy-associated mutation in the IL-4 receptor, Q576R, which was thought to be functionally important (41), does not affect IL-4 receptor signaling (42). In the case of mutations of the Fc{epsilon}RIß gene, the only published attempt at assessing the impact of mutations is a qualitative study looking at skin prick testing and in vitro Ag-induced basophil degranulation. It did not find any significant difference between nine 181L subjects and ten 181I subjects (43). However, for both tests used in this study, multiple factors could have masked an effect of the ß polymorphisms. For example, it has been long established that individuals can be classified as "low responder" or "high responder" based on the capacity of their basophils to respond to a challenge with IgE and anti-IgE (44). This difference in reactivity, the mechanism of which is unknown, could mask the effect of mutations in Fc{epsilon}RI. This demonstrates the need for a direct assessment of the potential role of the atopy-associated polymorphisms of the Fc{epsilon}RI ß-chain in atopy. We have performed such a study by testing the polymorphism effect on the two functions of ß, receptor expression amplification and signaling amplification.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Note added in proof.
 References
 
Cell culture, Abs, and reagents

Transfectants in the human monocytic cell line U937 were generated and maintained as described (32). Transfectants in the human basophil cell line KU812 cells were maintained in RPMI 1640 supplemented with 16% FBS, 2 mM L-glutamine, and 0.6 mg/ml G418. Anti-4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP)3 human IgE TAN was prepared from a hybridoma provided by Dr. Z. Eshhar (Weizman Institute of Science, Rehovot, Israel) as reported (45). Nonspecific human IgE was obtained from Calbiochem (La Jolla, CA).

Construction of stable transfectants

The mutated ß cDNAs (ßI181L-V183L and ßE237G) were generated from the wild-type (WT) ß cDNA subcloned in the vector pBJ1neo using the Quick-change kit (Stratagene, La Jolla, CA). The human {alpha} and {gamma} cDNAs were subcloned in the pCDL SR{alpha}296 eukaryotic expression vector (46), the human ß cDNA in the pBJ1neo eukaryotic expression vector containing a neo resistance cassette (47). U937 cells were cotransfected as described (32). When the {alpha} and {gamma}, but not ß, cDNAs were cotransfected, the empty pBJ1neo vector was cotransfected. U937 cells were cotransfected as described (32). Stable transfectants in the KU812 cells were generated by electroporation (300 V, 960 µF) with the {alpha} and {gamma} constructs and selected with G418.

Transient transfection

KU812 stable transfectants expressing Fc{epsilon}RI {alpha}{gamma}2 (5 x 106 cells) were cotransfected by electroporation with 10 µg of either a control construct, or one of the pBJ1neo ß constructs, and 1 µg of green fluorescent protein (GFP) construct (pGreen Lantern; Life Science, Bethesda, MD). Cells were analyzed 8–48 h after transfection. GFP fluorescence was measured by flow cytometry and used to gate transiently transfected cells.

Assessment of Fc{epsilon}RI expression by surface staining and flow cytometry

Cells were stained with 1 µg of biotinylated IgE, followed by streptavidin-PE (1:200) (PharMingen, San Diego, CA) and analyzed on a FACScalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Alternatively, cells were stained with anti-{alpha} mAb 15-1 followed by FITC-conjugated goat-anti mouse. Untransfected cells were processed in parallel and used as negative control.

Measurement of surface Fc{epsilon}RI expression by 125I IgE binding

The anti-NIP human IgE was iodinated with Iodogen (Pierce, Rockford, IL) following the manufacturer instructions. Bindability of iodinated IgE was checked. A binding curve in the presence of increasing concentrations of iodinated IgE was established and used to determine the saturating concentration of IgE after subtraction of nonspecific binding (48). Transfected cells were incubated with 125I IgE in saturating conditions, unbound IgE was removed, cell-associated counts were measured, and mean receptor numbers per cell were calculated from the binding data.

Immunoprecipitation and Western blotting

Cell lysis and immunoprecipitation were performed as previously described (32). Briefly, cells were lysed at a ratio of 3 x 107 cells/ml of lysis buffer (0.5% Triton X-100, 150 mM NaCl, 200 mM boric acid (pH 8.0), supplemented with the "complete" protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN). Where indicated, immunoprecipitates were treated with endo-ß-N-acetylglucosaminidase (endo-H) (New England Biolabs, Beverly, MA) as previously described (49). Samples were resolved on SDS 13% polyacrylamide gel, transferred to polyvinylidene difluoride membrane, and blotted with the Abs indicated. Immunoreactive proteins were visualized using alkaline phosphatase-coupled second-step reagents and enhanced chemifluorescence (Amersham, Arlington Heights, IL). Fluorescence was quantified using a Storm scanner (Molecular Dynamics, Sunnyvale, CA).

Measurement of calcium mobilization

U937 transfected cells were incubated with 2 µM fura-2 AM (Molecular Probes, Eugene, OR) and 0.2 mg/ml Pluronic (Molecular Probes) at room temperature for 30 min in a calcium (Ca2+) buffer containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, 10 mM Na HEPES (pH 7.4), 0.25 mM Sulfynpirazone (Sigma, St. Louis, MO) (50) and loaded with either 0.1–10 µg/ml or a saturating concentration (10 µg/ml) of anti-NIP IgE. When a nonsaturating concentration of anti-NIP IgE was used, nonspecific human IgE was added to a total concentration of 10 µg/ml. Cells were washed once in Ca2+ buffer. Intracellular Ca2+ concentrations were measured on bulk cell population at room temperature using a cuvette-based spectrofluorometer (Deltascan; Photon Technology, South Brunswick, NJ) before and after triggering with various concentrations (0.02–20 ng/ml) of the multivalent Ag NIP BSA (mean of 43 molecules of NIP per molecule of BSA). NIP BSA was prepared as described (45). Intracellular Ca2+ concentrations were calculated using the equation established by Grynkiewicz (51). Amplitudes of the initial Ca2+ response were calculated as the difference between the peak and the baseline.

Statistics

Averages are expressed ± SD. Comparisons between groups were performed with Student’s unpaired t test.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Note added in proof.
 References
 
The ß-chain is known so far to play two critical roles in Fc{epsilon}RI function. It amplifies surface Fc{epsilon}RI expression (31) and Fc{epsilon}RI signaling capacity (32, 33). We hypothesized that the association between the I181L-V183L and E237G polymorphisms and atopy could be due to an effect of these polymorphisms on either or both the amplifier functions of ß. To test this hypothesis, we have used a transfection system developed in our laboratory to study the signal amplifier function of ß. We have previously demonstrated that results obtained with this in vitro system parallel the results obtained in vivo with animal models of allergic reactions (32, 33). We have used this system to investigate the effect of the ß polymorphisms on receptor expression and signal amplification. We generated stable transfectants expressing Fc{epsilon}RI containing a WT ({alpha}ß{gamma}2) or variant ß ({alpha}ß181L-183L{gamma}2 or {alpha}ß237G{gamma}2) in the monocytic cell line U937, and analyzed the impact of these mutations on the two amplification functions of ß.

Effect of I181L-V183L and E237G mutations on the receptor expression amplification capacity of ß

Effect on Fc{epsilon}RI surface expression. Receptor expression was analyzed in the {alpha}ß{gamma}2, {alpha}ß181L-183L{gamma}2, and {alpha}ß237G{gamma}2 transfectants by flow cytometry after staining with labeled IgE. To accurately assess the whole spectrum of receptor expression, all of the selection-resistant clones were analyzed. (Fig. 1GoA). As expected given the receptor expression amplifier function of ß, {alpha}ßWT{gamma}2 clones express significantly more surface Fc{epsilon}RI than {alpha}{gamma}2 clones (31). With either ß variant, surface expression is amplified to an extent similar to that observed with WTß. To confirm this absence of effect of the ß polymorphisms on receptor expression, we analyzed the effect on receptor expression of transiently transfecting either WTß or one or the other of the ß variants in clones stably expressing {alpha}{gamma}2 (Fig. 1GoB). As expected, retransfection with WTß up-regulates receptor expression (31). Each ß variant was as efficient as WTß at up-regulating receptor expression. Therefore, it appears that these polymorphisms do not affect the capacity of ß to increase surface receptor expression.



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  		FIGURE 1. Distribution of Fc{epsilon}RI expression after transfection of the {alpha} and {gamma} cDNAs and constructs containing WTß, ßI181L-V183L, or ßE237G. A, U937 cells were stably transfected with the {alpha} and {gamma} cDNAs and with the empty pBJ1neo vector or with constructs containing either WTß, ßI181L-V183L, or ßE237G. Fc{epsilon}RI expression was analyzed on all the G418-resistant clones by flow cytometry with biotinylated human IgE followed by PE-conjugated streptavidin. Results are expressed as the mean fluorescence intensity (MFI) for each clone. Each dot represents one clone. The mean for each group is indicated by a white dot. The data for the {alpha}{gamma}2 and {alpha}ß{gamma}2 transfectants shown in open symbols are the same as in (31 ). B, A {alpha}{gamma}2-stable transfectant was transiently retransfected with either a control cDNA or a wild type, or variant ß cDNA, and with a GFP construct. Fc{epsilon}RI expression was measured on the GFP-positive cell population as in A, and followed up to 48 h after retransfection. Results are expressed as MFI of GFP-positive cells. For each type of transfectant MFI at each time point were compared with MFI at time 0. *, NS; #, p = 2.1E-3; +, p = 3.3 E-5. The data for the control and WTß retransfections shown in thin lines are the same as in (31 ).

 
Effect on Fc{epsilon}RI intracellular processing. Receptor expression at the cell surface is the result of a multistep process that includes protein synthesis of the three chains, assembly of the chains to form the trimer {alpha}{gamma}2 or the tetramer {alpha}ß{gamma}2, posttranslation modifications, for example, glycosylation of the {alpha}-chain, and export to the surface. We have recently shown that the ß-chain amplifies Fc{epsilon}RI expression by associating very early with the IgE-binding Fc{epsilon}RI {alpha}-chain and by promoting its intracellular processing (31). This effect of ß on {alpha} processing contributes to the increased receptor expression observed in the presence of ß. We have asked whether the ß polymorphisms could affect the capacity of ß to promote {alpha} processing. It is possible that a static analysis of surface receptor expression, such as the one we show in Fig. 1Go, could be unable to detect differences in efficiency in the steps that precede surface expression, and that, by limiting ourselves to an analysis of surface receptor expression, we could have missed an effect of the ß polymorphisms on intracellular receptor processing. To address this issue, we have compared the capacity of the ßI181L-V183L and ßE237G variants to that of the WTß to promote {alpha}-chain processing. The {alpha}-chain is highly and heterogeneously N-glycosylated, with ~50% of the molecular mass of cell surface {alpha} due to sugars. The newly synthesized {alpha}-chain is core glycosylated (mannose rich) in the endoplasmic reticulum and is thus sensitive to the action of endoglycosidase H (endo-H). It then is transferred to the Golgi, where terminal glycosylation occurs that replaces mannose rich sugars with complex sugars that are endo-H resistant. Thus, these various forms of {alpha} can be distinguished by a combination of endo-H treatment and SDS-PAGE (49). We have exploited {alpha}-chain glycosylation to follow processing of the receptor complex in the presence of the various forms of the ß-chain.

Transfected clones expressing the same levels of surface Fc{epsilon}RI and containing either the WTß or ßI181L-V183L, or ßE237G forms were lysed, and the {alpha}-chains were precipitated with an anti-{alpha} mAb (15-1), which can precipitate both mature and immature forms of {alpha}. The samples were then treated, or not, with endo-H and resolved by SDS-PAGE. Western analysis was performed using a polyclonal anti-{alpha} Ab (997) (data not shown) (31). A total of three to five clones and two to three uncloned cell populations of {alpha}ß{gamma}2, {alpha}ß181L-183L{gamma}2, and {alpha}ß237G{gamma}2 transfectants were studied. The bands corresponding to the mature and immature forms of {alpha} were identified on their m.w. and their sensitivity to endo-H, their intensities were measured by scanning densitometry, and the ratio between mature and immature {alpha} was calculated (Fig. 2Go). The amount of mature {alpha} for a given amount of immature {alpha} is not significantly different in the ßI181L-V183L- (4.8 ± 2) and the ßE237G (5.8 ± 1.5)-containing cells compared with the WTß-containing cells (4.9 ± 3.7). We conclude that the ß polymorphisms do not affect the capacity of ß to promote the processing of the {alpha}-chain.



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  		FIGURE 2. Fc{epsilon}RI {alpha} maturation and trafficking in the presence of WTß, ßI181L-V183L, or ßE237G. Cells were lysed and immunoprecipitated with the anti-{alpha} mAb 15-1. Immune complexes were treated or not with endo-H, subjected to SDS-PAGE and transferred to polyvinylidene difluoride membrane. Mature and immature forms of {alpha} were revealed by immunoblotting with anti-{alpha} polyclonal Ab 997. The bands corresponding to mature and immature {alpha} were scanned. A total of three to five clones and two to three uncloned transfected cell populations of {alpha}ß{gamma}2, {alpha}ß181L-183L{gamma}2, and {alpha}ß237G{gamma}2 transfectants were studied. Results are expressed as mean ± SD. The results for the {alpha}{gamma}2 and {alpha}ß{gamma}2 transfectants shown in thin lines include some data from (31 ).

 
Effect of I181L-V183L and E237G mutations on the signaling amplification capacity of ß

The ß-chain amplifies signals generated by the {gamma}-chain. As a result, in {alpha}ß{gamma}2 receptor-expressing cells’ signals are 3- to 5-fold more intense than those in {alpha}{gamma}2-expressing cells. We have investigated whether the ß polymorphisms affect the signal amplification capacity of ß. We have compared the signaling capacity of transfectants expressing Fc{epsilon}RI containing either a wild type, or one of the variant ß. Calcium flux was chosen as a readout of cell activation induced by Fc{epsilon}RI aggregation. Calcium flux is fundamental to the initiation and maintenance of an allergic response, and has been shown to correlate with degranulation (52), receptor phosphorylation, Syk activation, and in vivo responses (33). The amplitude of the initial calcium response depends on the number of receptors aggregated at the cell surface. To take this factor into account, eight {alpha}ß{gamma}2, eight {alpha}ß181L-183L{gamma}2, and six {alpha}ß237G{gamma}2 clones with mean receptor densities ranging from 6,000 to 90,000 receptors per cell were analyzed. In each phenotype the clones selected for analysis included low, medium, and high receptor expressers. Cells were loaded with the fluorescent Ca2+ dye fura-2 and saturated with the anti-NIP human IgE TAN. Intracellular Ca2+ concentrations were measured using a spectrofluorometer on bulk populations at baseline and after triggering with the multivalent Ag NIP BSA at an optimal concentration (20 ng/ml). Each clone was studied one to four times in independent experiments that all included at least one clone of each type. The amplitude of the initial calcium response was plotted as a function of Fc{epsilon}RI density (Fig. 3Go). At equal receptor density, there was no difference in the Ca2+ response after Fc{epsilon}RI triggering among clones expressing WTß or any of the ß variants.



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  		FIGURE 3. Amplitude of Ca2+ responses in transfectants expressing WTß, ßI181L-V183L, or ßE237G as a function of surface receptor density. Eight {alpha}ß{gamma}2, eight {alpha}ß181L-183L{gamma}2, and six {alpha}ß237G{gamma}2 clones with mean receptor densities ranging from 6,000 to 90,000 receptors per cell were loaded with the fluorescent Ca2+ dye fura-2 and human anti-NIP IgE (10 µg/ml) and triggered with an optimal concentration of the multivalent Ag NIP BSA (20 ng/ml). Each clone was studied one to four times in independent experiments. The amplitude of the initial Ca2+ response is plotted as a function of Fc{epsilon}RI density.

 
The possibility remains that a difference in signaling capacity between WTß and one of the ß variants could be detected when triggering is suboptimal, but not when it is optimal. To test this hypothesis we have performed a dose response of Ca2+ mobilization by varying the concentration of NIP BSA from 0.2 to 20 ng/ml. Fig. 4GoA shows the amplitude of the initial Ca2+ response for one WTß clone, one ßI181L-V183L clone, and one ßE237G clone, all expressing a mean of 30,000 receptors per cell. A total of three or four expression-matched clones from each type (mean receptors per cell = 12,000–75,000) were analyzed one to four times in independent experiments and showed comparable results (data not shown). No statistically significant difference could be detected between WTß, ßI181L-V183L, and ßE237G clones at any Ag concentration.



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  		FIGURE 4. Amplitude of Ca2+ responses in transfectants expressing WTß, ßI181L-V183L, or ßE237G as a function of triggering intensity. A, The experiment was performed as in Fig. 3Go except that one clone of each type expressing 30,000 receptors per cell was studied at various Ag (NIP BSA) concentrations. This figure is representative of a total of three independent experiments performed with the same clones. B, The experiments were performed as in A except that various concentrations of anti-NIP IgE were used for loading.

 
Another way to generate a suboptimal stimulus is to lower the concentration of the anti-NIP IgE used for cell loading, which results in subsaturating degrees of Fc{epsilon}RI occupancy. A saturating concentration of anti-NIP IgE (10 µg/ml) or lower, nonsaturating concentrations (0.5 and 2 µg/ml) were used. The total IgE concentration was kept at 10 µg/ml by addition of nonspecific human IgE. Fig. 4GoB shows the amplitude of the initial Ca2+ response as a function of IgE concentration for one WTß clone, one ßI181L-V183L clone, and one ßE237G clone, all expressing a mean of 30,000 receptors per cell. Again here, no difference could be detected between WTß, ßI181L-V183L, and ßE237G clones at any level of receptor occupancy.

We conclude that, despite their association with atopy, these ß polymorphisms appear to have no effect on Fc{epsilon}RI expression and function. Various explanations can be proposed for this discrepancy. These polymorphisms could affect unknown functions of ß. However, it is possible that there are still unknown functions of ß, some of which may be optimally detected in the physiological context of the receptor. Alternatively, these ß polymorphisms could be in linkage disequilibrium with unknown polymorphism(s) directly responsible for the atopic phenotype. These other polymorphisms could be in the ß gene or in other genes at the 11q13 locus, for example, HTm4, a molecule related to Fc{epsilon}RI ß (53). Other polymorphisms have been recently found in the regulatory and promoter regions of the ß gene, which may affect receptor expression, and whose effects on Fc{epsilon}RI expression and signaling have not been evaluated (54, 55, 56).


   Note added in proof.
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Note added in proof.
References
 
A paper published after acceptance of our manuscript (57) reported that the presence of ß181, ß183, or ß237 did not affect the signal amplification function of ß. This was assessed by ß-hexoseaminidase release, calcium mobilization, and secretion of IL-6, TNF-{alpha}, and LTC4 in bone marrow-derived mast cells from ß-/- mice reconstituted by retroviral infection.


   Footnotes
 
1 Current address: Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7627, Hospital Pitié Salpétrière, Paris, France. Back

2 Address correspondence and reprint requests to Dr. Jean-Pierre Kinet, Department of Pathology, RN227, Beth Israel Deaconess Medical Center, and Harvard Medical School, 99 Brookline Avenue, Boston, MA 02215. Back

3 Abbreviations used in this paper: NIP, 4-hydroxy-3-iodo-5-nitrophenylacetic acid; WT, wild type; GFP, green fluorescent protein; endo-H, endo-R-N-acetylglucosaminidase. Back

Received for publication April 20, 2000. Accepted for publication July 18, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Note added in proof.
 References
 

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